Recently an increased importance has been attached to clinical testing or diagnosis in which a patient's pathological condition or prognosis is judged or the dose of medicine to be administered to him is determined, by measuring the urinary or the blood level of hapten such as a low-molecular physiologically active substance which is present in his body and its metabolites or a medicine administered to him and its metabolites.
The haptens to be measured in this clinical testing or diagnosis include; androgens such as testosterone, dehydroepiandrosterone, androsterone; glucocorticoids such as cortisone, cortisol, corticosterone; mineral corticoids such as aldosterone; progestogens such as progesterone; estrogenes such as estriol, estradiol; thyroid hormones such as thyroxine, triiodothyronin; prostaglandin; physiological active amines such as L-dopa, epinephrine, norepinephrine, histamine and their metabolites.
The medicines to be measured in this testing or diagnosis include: medicines whose dose should be determined with utmost caution and whose effects have a correlation with their concentration in the blood or urine, i.e., digitalis preparations; antibiotics such as tetracyclin; psychotropic agents such as amphetamine; narcotic agents such as morphine; blood-coagulating agents or anticoagulating agents.
The term "hapten" used in the present specification means a low-molecular physiological active substance present in the human body and its metabolite or a medicine administered to the human body and its metabolite, which alone is not capable of producing an antibody and, only when bound with a substance which in itself is an antigen such as protein, polysaccharide, glycoprotein, is capable of producing an antibody, (hereafter to be referred to simply as a carrier) and is capable of reacting with said antibody produced.
Usually haptens occur in traces and they exist in complexed or conjugated form in blood or urine which are complex compositions. Thus an intricate, time-consuming process is needed for their detection and measurement.
Haptens may be measured by conventional methods such as the physico-chemical process, the immunochemical process, and the competitive protein binding process.
In the physico-chemical process, the hapten complex or conjugate in blood or urine is hydrolyzed by means of acid, alkali or enzyme and after purification, applied to chromatography, etc., for measurement. The operation involved, however, is complicated and time-consuming; in the course of hydrolysis steroids vanish through decomposition in the case of certain steroids or the greater part of the steroids are adsorbed in the course of chromatography; and in consequence the amount of steroid eluted is so small that it is not measurable.
Meanwhile, the immunochemical process is superior in the specificity of the reaction and the sensitivity of the measurement to said physico-chemical one; and at present numerous techniques of measuring trace hapten in the human body are practically available such as the agglutination inhibition reaction process and radioimmunoassay (to be abbreviated to RIA hereafter). In said agglutination inhibition reaction process, a hapten-carrier conjugate which represents the same hapten as the hapten to be measured, as bound with a carrier (hereafter to be referred to as ANTIGEN) is employed as the antigen. An antibody to the hapten to be measured (hereafter to be called the ANTIBODY) is obtained from an antiserum produced from mammals such as guinea pigs, rabbits or sheep which have been immunized with the ANTIGEN. Then using said ANTIBODY and an ANTIGEN-sensitized particle which has been obtained by sensitizing blood cells or a fine particle of high-molecular latex (hereafter to be called particle), the agglutination reaction occurs between the two components. In this process the agglutination reaction inhibited by the presence of the hapten to be measured is utilized to obtain this measurement. Unlike said physico-chemical process, this is an extremely simple process which can measure the hapten existing as a complex or conjugate in the blood or urine without complicated operations such as hydrolysis or chromatography. The sensitivity of this process is about 100 ng/ml even when blood cells, i.e., an excellent particle or sensitizing ANTIGEN is adopted. Since most haptens which are known to be worth measuring in the human body occur in the range of 500 pg/ml-50 ng/ml, they have to be concentrated for successful measurement.
In the competitive protein binding process, instead of the antibody employed in RIA a hapten receptor or a binding protein present in the human body is employed. With a measured value well correlated to the biological activity exhibited by hapten, this process is found useful, but it has the drawbacks that only a few haptens such as estrogen, cortcosteroid, thyroid hormone have known receptors; but a complicated operation is needed for extraction and purification of these receptors and these receptors after extraction do not keep long.